Abstract

Regulation of energy metabolism is critical for the prevention of obesity, diabetes, and hepatic steatosis. Here, we report
an important role for the pleckstrin homology–related domain family member, T-cell death–associated gene 51 (TDAG51), in the regulation of energy metabolism. TDAG51 expression was examined during adipocyte differentiation. Adipogenic potential
of preadipocytes with knockdown or absence of TDAG51 was assessed. Weight gain, insulin sensitivity, metabolic rate, and liver lipid content were also compared between TDAG51-deficient
(TDAG51−/−) and wild-type mice. In addition to its relatively high expression in liver, TDAG51 was also present in white adipose tissue
(WAT). TDAG51 was downregulated during adipogenesis, and TDAG51−/− preadipocytes exhibited greater lipogenic potential. TDAG51−/− mice fed a chow diet exhibited greater body and WAT mass, had reduced energy expenditure, displayed mature-onset insulin
resistance (IR), and were predisposed to hepatic steatosis. TDAG51−/− mice had increased hepatic triglycerides and SREBP-1 target gene expression. Furthermore, TDAG51 expression was inversely
correlated with fatty liver in multiple mouse models of hepatic steatosis. Taken together, our findings suggest that TDAG51
is involved in energy homeostasis at least in part by regulating lipogenesis in liver and WAT, and hence, may constitute a
novel therapeutic target for the treatment of obesity and IR.

With a dramatic rise in the prevalence of obesity and type 2 diabetes worldwide, there is an urgent need to better understand
the genes involved in the metabolic processes underlying these conditions. Obesity results from an increase in white adipose
tissue (WAT) mass, which occurs as a result of two processes, adipocyte hypertrophy and hyperplasia. The former process indicates
an increase in adipocyte size, and the latter is the result of an increase in mature adipocyte number through preadipocyte
recruitment and adipocyte differentiation (1,2). The transcription factors peroxisome proliferator–activated receptor γ (PPARγ) (3,4) and CCAAT/enhancer–binding protein-α (C/EBPα) (5–7) play a crucial role during adipogenesis. In the later stages of terminal differentiation, proteins such as adipocyte-specific
fatty acid–binding protein (aP2), adiponectin, and leptin are expressed (8). A fine balance between adipocyte hypertrophy and adipogenesis exists to prevent the formation of dysfunctional adipose
tissue, since large cells are more likely to be insulin resistant (9,10) and therefore can influence adipose tissue metabolism. Despite significant progress over the last few years, additional
genes and factors that influence adipogenesis, adipose tissue metabolism, and ultimately energy homeostasis still remain to
be uncovered.

A recent microarray analysis of differentiating 3T3-L1 preadipocytes found T-cell death associated gene 51 (TDAG51), among other genes, to be induced within the first 8 h of adipogenesis (11) and was categorized in the same gene cluster as C/EBPβ and C/EBPδ, early adipogenic genes crucial for initiating the adipogenic gene expression cascade (12). Given the importance of the first 24 h in determining preadipocyte cell fate (13,14), this finding suggests that TDAG51 may be a novel candidate gene involved in adipogenesis. TDAG51 is a member of the pleckstrin homology (PH)–like domain family
(human homolog PHLDA1) (15) and was first demonstrated to be induced upon T-cell activation–mediated apoptosis in culture (16). However, in contrast to these in vitro findings, TDAG51-deficient (TDAG51−/−) mice displayed no apparent defects in T-cell apoptosis, number, or function (17). In addition to its PH-like domain, TDAG51 possesses distinct COOH-terminal proline-glutamine and proline-histidine repeats
(18), which may be involved in its proapoptotic function in some cell types (19). TDAG51 has been suggested to play a role in tumorigenesis (20,21), and we have previously shown its expression in atherosclerotic lesions from hyperhomocysteinemic mice (22), although the precise physiological role of TDAG51 is not well understood. To date, the role of TDAG51 in adipogenesis and
energy metabolism has not been examined.

We evaluated the protein expression pattern of TDAG51 in several tissues of wild-type (WT) C57BL/6 mice, which indicated that
in addition to liver, which expresses the highest levels of TDAG51, WAT also expresses TDAG51. Here we report that TDAG51
expression was modulated during adipogenesis and that TDAG51−/− preadipocytes exhibited greater lipid accumulation under adipogenic conditions as compared with WT cells. TDAG51−/− mice displayed an age-related increase in whole-body adiposity and an overall metabolic profile resembling the metabolic
syndrome. Furthermore, TDAG51 expression was inversely correlated with fatty liver in several mouse models of hepatic steatosis.
These findings suggest a previously unknown function for TDAG51 in promoting negative energy balance by regulating adipocyte
and hepatic lipogenesis, thereby favoring energy expenditure.

Cell culture.

Mouse 3T3-L1 cells were cultured, differentiated, and stained with Oil Red O as previously described (23). Pooled protein extracts were obtained from isolated human preadipocytes and their corresponding differentiated adipocytes
from four lean female subjects (Zenbio).

mRNA quantification by real-time PCR.

Total RNA was isolated using the RNeasy kit (Qiagen) according to the manufacturer’s protocol. Using a High-Capacity cDNA
Reverse Transcription Kit (Applied Biosystems), total RNA was reverse transcribed to obtain cDNA. Quantitative real time-PCR
(qRT-PCR) was performed using Fast SYBR Green PCR Master Mix (Applied Biosystems) in the AB7900 HT Fast Real-Time PCR System.
For a list of primer sequences see Supplementary Table 1. Data analysis was performed using the ΔΔC(T) method and normalized to 18S, unless otherwise indicated.

Isolation and culturing of primary cells.

Primary preadipocytes were cultured by isolating the stromal-vascular fraction from mouse WAT. In brief, WAT was minced, collagenase
(Sigma) digested, and centrifuged to separate the stromal-vascular fraction from the mature adipocyte fraction. The pelleted
cells were then plated and induced to differentiate in Dulbecco’s modified Eagle’s medium/F12 containing 2% FBS, 1 μg/mL insulin,
250 nmol/L dexamethasone, 0.5 mmol/L isobutylmethylxanthine, 60 μmol/L indomethacin, and 2.5 μmol/L rosiglitazone. Cells were
stained with Oil Red O as previously described (23), or protein lysates were collected for Western blotting. Primary hepatocytes isolated from the liver were plated on collagen-coated
six-well plates (BD) and allowed to attach for 2–3 h in Williams’ medium E (Invitrogen), after which medium was replaced with
Williams’ medium E containing 10% FBS. Isolated hepatocytes were incubated overnight at 37°C and the experiments were performed
the following day.

Mice.

TDAG51−/− mice were generated previously (17) and back-crossed onto the C57BL/6 background for at least nine generations and genotyped as described in the Supplementary Data. C57BL/6 or TDAG51−/− mice were maintained under 12-h light-dark cycles with unlimited access to standard rodent chow and water. At 8 weeks of
age, male WT or TDAG51−/− mice (n = 8) were either continued on the regular chow diet or placed on a high-fat diet (42% of kcal from fat, TD.88137; Harlan
Teklad). At 28 weeks of age, mice were fasted for 14 h before they were killed by cervical dislocation under anesthesia. Blood
was drawn from the heart, and tissues were immediately collected for histology or biochemical assessment. Only male mice were
used in this study. Unless stated otherwise, only chow diet–fed mice were examined. All protocols for animal use and euthanasia
were approved by the McMaster University Animal Research Ethics Board.

Computed tomography and positron emission tomography.

WT and TDAG51−/− mice were fasted overnight and given an intravenous injection of 2-(18F) fluoro-2-deoxyglucose (2-FDG). The mice were kept under heat lamps for 30 min, anesthetized using isoflurane, and scanned
on a small animal positron emission tomography (PET) scanner for visualization of glucose uptake. The mice were placed in
a single-photon emission computed tomography (SPECT)/CT scanner for determination of body fat content (24).

Cytometric inflammatory bead array.

Plasma cytokines were measured using a bead cytometric array (BD) according to the manufacturer’s protocol.

Metabolic studies.

For glucose tolerance tests (GTTs), mice were fasted overnight and given an intraperitoneal injection of glucose (1.5 mg/g
body weight). For insulin tolerance tests (ITTs), mice were fasted for 6 h and intraperitoneally injected with 0.75 units/kg
of insulin. Blood was collected from the tail vein, and glucose concentrations were measured using a hand-held glucometer.
For in vivo insulin signaling experiments, tissues were collected 15 min after an intraperitoneal injection of insulin (0.75
units/kg) in fasted mice as previously described (25). Metabolic studies using indirect calorimetry were performed using the Columbus Laboratory Animal Monitoring System (CLAMS)
as described previously (26). Core body temperature was determined in the CLAMS after subcutaneous implantation, under isoflurane anesthesia, of minimiter
probes (Respironics, Inc.) in the dorsal subscapular region.

Transmission electron microscopy.

Interscapular brown adipose tissue (BAT) was excised, cut into 2-mm3 pieces, and immersed in 2% glutaraldehyde in cacodylate buffer. Tissues were processed using standard protocols and embedded
in Spurr resin. Ultrathin sections were stained with uranyl acetate and lead citrate and observed under a JEOL 200CX TEMSCAN
microscope.

Hepatic TG content and VLDL-TG secretion.

Approximately equivalent weights of frozen liver tissue from mice were homogenized in PBS, and TGs were extracted using the
Folch extraction method as previously described (27). Hepatic VLDL-TG secretion was measured as previously described (28). In brief, fasted 8-week-old mice were injected intraperitoneally with 1 g/kg poloxamer-407 dissolved in saline, and blood
samples were collected for serum TG measurements at various time points.

Hepatocyte de novo lipogenesis and palmitate oxidation.

For determination of de novo lipogenesis, hepatocytes were incubated with media containing sodium acetate (5 mmol/L) and 3H-acetate (1 µCi/mL; Perkin Elmer) for 1 or 4 h in the presence or absence of 10 nmol/L insulin. Cells were washed twice with
PBS, and lipids were extracted by the method of Bligh and Dyer (29). Neutral lipids were quantified as previously described (30). Lipid bands were visualized and scraped into counting vials for measuring radioactivity. Palmitate oxidation was determined
as described previously (31). In brief, sodium palmitate (0.5 mmol/L) and 14C-palmitate (0.5 µCi/mL; Perkin Elmer) were allowed to conjugate to 2% BSA for 2–3 h and then incubated with primary hepatocytes
for 4 h, in the presence or absence of 10 nmol/L insulin. Radiolabeled CO2 was liberated by adding an equal volume of 1 mol/L acetic acid and trapped by the addition of benzethonium hydroxide (1 mol/L)
over 90 min. The tube containing trapped 14CO2 was placed in a scintillation vial and counted.

Statistical analysis.

Data are expressed as the mean ± SEM unless otherwise indicated. Statistical analyses were performed using unpaired, two-tailed
Student t test, and significance was defined as P < 0.05.

RESULTS

TDAG51 is expressed in preadipocytes and is downregulated during adipogenesis.

TDAG51 protein expression was examined in various tissues from adult C57BL/6 mice. In addition to the liver and lung, which
have abundant TDAG51 levels at the mRNA and protein level (15,17), WAT also expressed TDAG51 protein (Fig. 1A). Previous microarray analysis during adipogenesis indicated that TDAG51 mRNA expression is modulated in early adipogenesis (11). Therefore, we carried out qRT-PCR analysis, which confirmed that TDAG51 mRNA was significantly induced in the first 2 h of adipogenesis followed by a sharp decline back to baseline expression (Fig. 1B). We next sought to evaluate the expression profile of TDAG51 at the protein level during adipocyte differentiation. TDAG51
protein expression was induced in the first 4 days poststimulation, followed by a gradual decline after day 4, and diminished
expression by day 14 of differentiation, when the vast majority of cells had accumulated large lipid droplets, as shown by
Oil Red O staining (Fig. 1C, top). In addition, TDAG51 expression showed an inverse correlation with PPARγ expression during adipogenesis (Fig. 1C).

TDAG51 is expressed in preadipocytes and is downregulated during adipogenesis. A: Western blotting for TDAG51 using whole-tissue lysates from WT C57BL/6 mice. B: qRT-PCR for TDAG51 mRNA expression in 3T3-L1 preadipocytes during differentiation. Data represent the mean ± SD. **P < 0.01 vs. day 0 cells. C: 3T3-L1 cells were induced to differentiate for 14 days and stained with Oil Red O to detect neutral lipids (top). Total lysates were assessed for PPARγ, TDAG51, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control) by
immunoblotting (bottom). D: Mouse embryonic fibroblasts (MEFs) isolated from WT C57BL/6 mice were induced to differentiate (at passage 5), and protein
extracts were collected and subjected to immunoblotting. β-Actin was used as a loading control. E: TDAG51, perilipin (positive control for mature adipocytes), and β-actin protein expression in human preadipocyte and corresponding
mature adipocyte protein extracts (pooled from four subjects). F: 3T3-L1 cells on day 1 of differentiation were treated with 30 μg/mL cycloheximide, and protein lysates were collected at
the indicated time points. TDAG51, PPARγ, C/EBPβ, and β-actin expression were assessed through immunoblotting. (A high-quality
color representation of this figure is available in the online issue.)

In support of the findings in 3T3-L1 cells, an inverse correlation between TDAG51 and PPARγ expression and/or adipogenesis
was also observed in mouse embryonic fibroblasts induced to differentiate into adipocytes (Fig. 1D). We next examined protein extracts from pooled human preadipocytes and their corresponding differentiated mature adipocytes,
which also showed disappearance of TDAG51 in mature adipocytes (Fig. 1E). These findings indicate that TDAG51 is downregulated in differentiated lipid-laden adipocytes, suggesting that TDAG51 may
play an important role in adipogenesis.

To reconcile the temporal difference in the expression of TDAG51 mRNA and protein during adipogenesis, 3T3-L1 cells were treated
with cycloheximide to block protein translation. As shown in Fig. 1F, relative to other proteins that are crucial for adipogenesis, such as PPARγ and C/EBPβ, TDAG51 protein is considerably more
stable.

In order to examine the role of TDAG51 in adipogenesis, primary stromal-vascular cells (SVCs) were isolated from WT and TDAG51−/− WAT and induced to differentiate. TDAG51−/− SVCs exhibited earlier and/or greater expression of adipogenic markers such as PPARγ2, aP2, FAS, perilipin, and C/EBPα on
day 2 (Fig. 2A). By day 5, most of these adipogenic markers reached similar levels in WT and TDAG51−/− SVCs. However, despite the plateau in expression of these adipogenic markers, TDAG51−/− SVCs exhibited increased lipid accumulation, as determined by Oil Red O staining (Fig. 2B), and enhanced adiponectin secretion, a marker of mature adipocytes (Fig. 2C).

Absence of TDAG51 enhances lipid accumulation in adipocytes. A: Immunoblotting for adipogenic markers on day 0, 2, 3, 4, and 5 of WT or TDAG51−/− primary preadipocyte differentiation. B: Preadipocytes on day 8 were stained with Oil Red O and representative images were taken (top). The staining was quantified (bottom) (n = 3). C: Adiponectin secretion from primary preadipocytes (n = 5). Data are expressed as the mean ± SD. *P < 0.05 and **P < 0.01 vs. WT on the same day of differentiation. (A high-quality color representation of this figure is available in the
online issue.)

To complement these findings, both silencing/short interfering (si)RNA and short hairpin (sh)RNA targeting mouse TDAG51 were used to knockdown TDAG51 expression in 3T3-L1 cells. Similar to TDAG51−/− SVCs, siTDAG51-transfected cells had significantly greater lipid accumulation compared with siControl-transfected cells (Supplementary Fig. 1A and B). An shTDAG51 construct was used, leading to a stable 30% knockdown in TDAG51 mRNA expression (Supplementary Fig. 1C). Even this modest decrease in mRNA expression led to a striking decrease in TDAG51 protein (Supplementary Fig. 1D) and resulted in significantly greater adiponectin secretion from shTDAG51 cells on day 3 (Supplementary Fig. 1E). Expression of C/EBPδ and C/EBPβ mRNA was higher within the first 8 h of differentiation but dropped off more quickly, and similarly mRNA expression of preadipocyte
factor-1 (Pref-1) declined more quickly in shTDAG51 cells on day 1 as compared with shControl cells (Supplementary Fig. 1F). Analysis of late markers of adipogenesis, such as expression of PPARγ, aP2, and C/EBPα mRNA, indicated that they were expressed earlier (day 2) and to a greater extent in shTDAG51 cells (Supplementary Fig. 1G).

To study the effect of loss of TDAG51 on weight regulation in vivo, 8-week-old chow diet–fed male TDAG51−/− mice were monitored over 20 weeks and compared with age- and sex-matched heterozygous TDAG51+/− and WT mice. Body weight was not significantly different between genotypes at 8 weeks of age (Fig. 3A and Supplementary Fig. 2). However, over time, in comparison with WT mice, TDAG51−/− mice exhibited a significant increase in body weight (Fig. 3A), which was not observed for TDAG51+/− mice (Supplementary Fig. 2). There was no significant difference in tissue weights (Supplementary Fig. 3A and B) except for epididymal WAT (eWAT), where a significant increase was observed in TDAG51−/− mice (Fig. 3B). CT scans in 28-week-old mice showed that TDAG51−/− mice have a fivefold increase in total body WAT (Fig. 3C), with fat deposition in both the visceral and subcutaneous depots (Fig. 3C, top). A significant increase in adiposity was also observed in the bone marrow of TDAG51−/− mice (Supplementary Fig. 4A and B). Histological examination of the eWAT revealed that TDAG51−/− adipocytes were significantly enlarged compared with WT controls (Fig. 3D). In support of enhanced adipogenic and lipogenic potential observed in cultured preadipocytes (Fig. 2A–C), mRNA expression of several adipogenic genes (aP2, PPARγ, and C/EBPβ) was significantly increased in WAT from TDAG51−/− mice (Fig. 3E). These findings demonstrate that absence of TDAG51 enhances adipocyte lipid accumulation, leading to WAT expansion and increased
body mass in TDAG51−/− mice.

We next examined metabolic parameters using indirect calorimetry. Food consumption in WT and TDAG51−/− mice was not significantly different (Fig. 4A), and although there was a trend toward lower locomotor activity in TDAG51−/− mice, the difference did not reach statistical significance (Fig. 4B). However, TDAG51−/− mice exhibited a decrease in metabolic rate as indicated by a significant reduction in oxygen consumption (Vo2) during both day and night hours (Fig. 4C). Core body temperature was also lower in TDAG51−/− mice during the night, which corresponded with their lower Vo2 (Fig. 4D).

We explored whether changes in BAT, an important regulator of thermogenesis in mice, could explain the difference in energy
expenditure. Interscapular BAT from WT and TDAG51−/− mice was examined to assess cell morphology, and we found an overall increase in lipid droplet size in BAT from TDAG51−/− mice (Fig. 4E). We next examined expression of thermogenic and mitochondrial genes in BAT from WT and TDAG51−/− mice. No significant differences were found in the expression of UCP1, CIDEA, PGC1α, CPT1b, or PPARγ, and we did not detect any differences in the expression of components of the electron transport chain (Supplementary Fig. 6A). Furthermore, no overall difference in UCP-1 immunostaining of BAT paraffin sections was observed between genotypes (Supplementary Fig. 6B). To assess mitochondrial morphology and size, BAT was further examined using transmission electron microscopy; however,
no striking differences in mitochondria shape, size, or cristae were observed between genotypes (Supplementary Fig. 6C). Given that TDAG51 expression is ∼90% lower in BAT compared with WAT of WT mice (Supplementary Fig. 6D), it is possible that BAT function in TDAG51−/− mice would not be directly affected by the absence of TDAG51. These findings suggest an increase in ectopic lipid deposition
in BAT; however, BAT function does not appear to be significantly affected.

TDAG51−/− mice develop age-associated insulin resistance.

Fasting plasma glucose and insulin levels were significantly elevated in TDAG51−/− mice at 28 weeks of age (Fig. 5A and B). In young 8-week-old WT and TDAG51−/− mice, an age where whole-body, liver, and WAT weights were indistinguishable (Fig. 3A and Supplementary Fig. 7A), GTTs and ITTs revealed no differences between genotypes (Supplementary Fig. 7B and C). However, at 28 weeks of age, when TDAG51−/− mice exhibited a significant increase in WAT and body weight, TDAG51−/− mice were also significantly more glucose intolerant (Fig. 5C) and insulin resistant (Fig. 5D) compared with WT mice. The efficiency of glucose uptake in various tissues was examined using PET scanning, which revealed
a significant decrease in glucose uptake in skeletal muscles and WAT of TDAG51−/− mice (Fig. 5E). WAT from TDAG51−/− mice also showed decreased insulin receptor signaling, as indicated by reduced tyrosine (Y608) phosphorylation of IRS-1 and
increased phosphorylation on serine (S612), which inhibits insulin receptor signaling (Fig. 5F). However, at least within this time interval, insulin-stimulated Akt phosphorylation and SREBP-1 activation were similar
in WAT from WT and TDAG51−/− mice. Consistent with the presence of increased adiposity, decreased glucose disposal, and insulin resistance (IR), plasma
FFAs were also significantly elevated in TDAG51−/− mice (Table 1).

TDAG51−/− mice develop age-associated IR. Fasting glucose (A) and fasting insulin (B) concentrations in 28-week-old male mice (n = 7–8). C: GTT after an overnight fast and the area under the curve (AUC) in the right panel (n = 4). D: ITT after a 6-h fast and the AUC (right) (n = 6–7). E: Intravenous injection of 2-FDG followed by a PET scan. Quantification of 2-FDG in various tissues (n = 5). PET scan images from representative mice are shown in the right panel. F: Mice were injected with insulin after a 6-h fast, and eWAT was collected 15 min later for Western blotting examining insulin
receptor signaling (n = 5 mice). G: Pancreatic islets taken at the same magnification, where red staining represents insulin and green depicts glucagon. Data
are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01 vs. WT.

To investigate the cause of hyperinsulinemia and IR in TDAG51−/− mice, pancreatic islets were histologically assessed using insulin and glucagon staining. No discernible differences in islet
size or shape were observed between genotypes (Fig. 5G). Overall, these findings demonstrate that TDAG51−/− mice display an age-related impairment in insulin sensitivity that is likely related to increased adiposity in these mice.

Given the high levels of TDAG51 expression in the liver, we sought to determine if TDAG51 expression was altered in fatty
livers by examining three mouse models of nonalcoholic fatty liver disease. Dietary, genetic, and chemically induced steatosis
were all highly correlated with reduced TDAG51 protein expression (Fig. 6A). Interestingly, absence of TDAG51 increased hepatic lipid accumulation and steatosis in 28-week-old mice on chow diet, as
determined by histological staining and biochemical measurement of liver TG (Fig. 6B and C), a phenotype that was exacerbated in TDAG51−/− mice fed a high-fat diet for 20 weeks (Fig. 6B). Given that significant metabolic differences existed between genotypes on a standard chow diet, only chow diet–fed mice
were further examined. To rule out the possibility that loss of TDAG51 resulted in hepatic TG accumulation due to impaired
hepatic VLDL-TG secretion, serum TG concentrations were measured over the course of 4 h after injection of poloxamer-407,
an inhibitor of lipoprotein lipase. There were no significant differences in the rate of hepatic TG secretion (Fig. 6D).

TDAG51−/− livers showed increased staining for proteins covalently adducted with 4-hydroxynonenal, a marker of lipid peroxidation (Fig. 6E), and an increased number of granulomas indicative of inflammation (Fig. 6F), both of which are consistent with greater steatosis. However, there were no signs of hepatocellular injury as measured
by plasma ALT (Table 1), and hepatic cholesterol content was not affected (data not shown). In agreement with increased hepatic TG in TDAG51−/− livers, there was a marked increase in insulin-stimulated SREBP-1 activation, a master regulator of hepatic lipogenesis (32–34), as indicated by reduced expression of the precursor form of SREBP-1 (p125) and enhanced expression of the mature form (p68)
(Fig. 6G), which was not affected under basal fasted conditions (Supplementary Fig. 8A). Insulin-injected TDAG51−/− livers also had reduced serine 473 phosphorylation of Akt, suggestive of hepatic IR (Fig. 6G). Expression of lipogenic enzymes and SREBP-1 target genes, FAS and stearoyl-CoA desaturase-1 (Scd-1), were induced in livers
from TDAG51−/− mice under basal fasting conditions (Fig. 6H). Furthermore, preliminary microarray gene expression analysis of liver from chow diet–fed WT and TDAG51−/− mice (K.N.M., R.C.A., unpublished data) revealed an increase in >10 SREBP target genes in TDAG51−/− livers (Supplementary Table 3).

To determine whether these results were secondary to the insulin-resistant phenotype or intrinsic to the loss of TDAG51, primary
hepatocytes from 6-week-old WT and TDAG51−/− mice, which displayed no differences in body weight or metabolic phenotype, were examined. TDAG51−/− hepatocytes responded normally to insulin (Supplementary Fig. 8B) but had enhanced basal and insulin-stimulated lipogenesis (rate of newly synthesized fatty acid incorporation into TG) (Fig. 7A). Palmitate oxidation, on the other hand, was not different between genotypes when examined basally or upon insulin stimulation
in primary hepatocytes (Fig. 7B). Furthermore, levels of PPARα, a transcription factor involved in fatty acid catabolism, was not affected by the absence
of TDAG51 in liver tissue (Supplementary Fig. 8A). Consistent with enhanced de novo lipogenesis, primary hepatocytes from TDAG51−/− mice expressed higher mRNA levels of the SREBP-1c target genes glucokinase (GK) and acetyl CoA carboxylase 1 (ACC1) (Fig. 7C). These results in cultured hepatocytes are in full agreement with our findings in livers from TDAG51−/− mice, suggesting that deletion of TDAG51 promotes lipogenesis at least in part through increased SREBP-1 activation and its downstream target gene expression.

DISCUSSION

Dysregulation of the pathways involved in adipogenesis, lipid metabolism, and energy homeostasis can lead to the development
of obesity, hepatic steatosis, diabetes, and dyslipidemia. Here, we present novel evidence that TDAG51 plays a significant
role in the regulation of energy and lipid metabolism. First, the findings of this study show for the first time that TDAG51
is expressed in WAT and that its protein expression is modulated during adipogenesis. Given that TDAG51 protein expression
is minimal or absent in mature mouse and human adipocytes, TDAG51 may be a useful preadipocyte marker. Second, deletion of
TDAG51 enhances lipid accumulation during adipogenesis and accelerates fatty acid/TG synthesis in primary hepatocytes. Third,
the absence of TDAG51 leads to an age-associated increase in whole-body adiposity and hepatic TG accumulation, and decreases
metabolic rate and insulin sensitivity in standard chow diet–fed WT mice. Notably, the phenotype observed in TDAG51−/− mice resembles several aspects of high-fat diet–fed WT mice. Finally, TDAG51 protein expression is inversely correlated with
hepatic steatosis.

The age-dependent increase in adiposity, hepatic TG accumulation, and systemic IR in TDAG51−/− mice are suggestive of a chronic and cumulative effect, likely attributable to a decrease in metabolic rate and increased
lipogenesis. Excessive accumulation of TG can culminate in the expansion of WAT, ectopic lipid accumulation, and eventually
IR in combination with chronic inflammation and oxidative stress (35,36), which is consistent with the phenotype observed in older TDAG51−/− mice. Moreover, when cells become insulin resistant, not all pathways are blunted as lipogenic pathways remain active, a
concept known as selective IR (37). The reduced Akt phosphorylation in combination with increased SREBP-1 activation seen in livers from insulin-injected TDAG51−/− mice is consistent with selective IR. The absence of TDAG51 in mice culminated in increased TG deposition in the liver. Numerous
studies show a strong correlation between fatty liver and whole-body IR (38–42), suggesting that increased hepatic steatosis and selective IR in the liver of TDAG51−/− mice may be contributing to the systemic IR phenotype in these mice. Given that SREBP-1 is a master regulator of hepatic
de novo lipogenesis (32–34) and an increase in SREBP-1 target gene expression was observed in TDAG51−/− livers, the absence of TDAG51 likely leads to hepatic steatosis through increased SREBP-1 activity. Consistent with this
result, primary TDAG51−/− hepatocytes exhibited a significant enhancement in the rate of lipogenesis, which correlates with the strong association
between TDAG51 reduction and fatty livers. Taken together, these data suggest a role for TDAG51 in the negative regulation
of SREBP-mediated lipogenesis and prevention of fatty liver.

TDAG51 has several important domains, including a PH-like domain, which appears to be a likely candidate for functional significance
in the context of cell signaling and metabolism. For instance, a recent study identified a novel PH domain–containing protein
involved in the regulation of adipocyte insulin signaling (43). Indeed, TDAG51 was identified as a specific IGF-1 target gene in NIH-3T3 fibroblasts and was shown to mediate the effects
of IGF-1 on cell survival (44). Furthermore, previous studies suggest that extracellular signal–related kinase (ERK) and p38 mitogen-activated protein
kinase (p38 MAPK) signaling can modulate TDAG51 expression (44,45). However, the exact cellular mechanisms by which IGF-1, ERK, or p38 MAPK induces TDAG51 expression are unknown.

PH domains play an important role in recruiting target proteins to cell membranes through their binding interactions with
phospholipids such as phosphatidylinositol (3-5)-triphosphate (PIP3) and phosphatidylinositol (4,5)-bisphosphate (PIP2) (46,47). Interestingly, TDAG51 has a high stringency PIP3-binding site in its PH-like domain, as predicted by ScanSite (48). Furthermore, the PH-like domain of TDAG51 exhibits a high degree of homology to that of phosphatidylinositol 3-kinase (PI3K)
enhancer (PIKE), as predicted by the Protein Homology/Analog Y Recognition Engine (Phyre2) (49). Interestingly, PIKE has recently been implicated in playing a role in adipogenesis, WAT regulation, and insulin sensitivity,
and in contrast to TDAG51−/− mice, PIKE−/− mice are protected from diet-induced weight gain and IR (50). Thus, we are currently exploring whether the PH-like domain of TDAG51 is indeed mediating its effects on the regulation
of lipogenesis and whole-body energy metabolism and whether the PI3K/Akt pathway may be involved.

Collectively, the results of our study illustrate that TDAG51 plays an important role as a regulator of carbohydrate and lipid
metabolism, at least in part through regulating SREBP activity, which may contribute to the phenotypes observed with the metabolic
syndrome. Understanding this newly identified cellular factor in lipid metabolism and adipogenesis as well as its precise
regulation under normal and pathophysiological conditions will be crucial for the development of effective treatments against
diabetes and obesity.

ACKNOWLEDGMENTS

R.C.A. and G.R.S. are supported by grants from the Heart and Stroke Foundation of Ontario (NA-6024) and the Canadian Institutes
of Health Research (CIHR) (MOP-111239 and MOP-67116). Financial support from St. Joseph’s Healthcare Hamilton is also acknowledged.
K.N.M. is supported by National Institute of Diabetes and Digestive and Kidney Diseases grant R01-DK-074487-01. S.B. is supported
by a Heart and Stroke Foundation of Canada Doctoral Award. M.D.F. is a CIHR Banting Postdoctoral Fellow. G.R.S. is a Canada
Research Chair in Metabolism and Obesity. R.C.A. is a Career Investigator of the Heart and Stroke Foundation of Ontario (CI-7408)
and holds the Amgen Canada Research Chair in Nephrology.

No potential conflicts of interest relevant to this article were reported.

S.B. performed experiments, collected and interpreted data, and wrote the manuscript. Š.L., M.D.F., R.P., and E.G.L. performed
experiments, contributed to data interpretation, and edited the manuscript. H.J. and R.J.F. assisted with experiments. K.N.M.,
G.R.S., and R.C.A. contributed to data interpretation and discussion and reviewed the manuscript. R.C.A. is the guarantor
of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.

The authors thank Dr. Y. Choi (University of Pennsylvania, Philadelphia, PA) for kindly providing the TDAG51+/− mice. The authors acknowledge the help of K. Karimi (McMaster University) with the BD cytometric bead array and are grateful
to C. Saab and R. Rhem (McMaster University) for their technical assistance with the animal imaging.

. Apoptosis and insulin resistance in liver and peripheral tissues of morbidly obese patients is associated with different stages
of non-alcoholic fatty liver disease. Diabetologia2011;54:1788–1798pmid:21455726